Homologous genetic recombination (sometimes called general recombination) is a naturally occuring process in many cells. While the actual mechanisms mediating this process are not firmly established, it involves the exchange of equivalent lengths of single stranded DNA between two interacting double stranded DNA segments. In the Holliday model of homologous recombination (named for Robin Holliday, who proposed the model in 1964) two similar, double stranded DNA segments (called duplexes) align with one another. After this, one single strand in each duplex breaks, and its free end invades the other duplex and ligates to the remaining end of the other broken strand. The resulting molecule is called a Holliday intermediate, and the location where strands cross over into opposite duplexes is known as a branch. In the final step of this process, the Holliday intermediate is cut in the region of the branch, and the cut ends are ligated to form two separate recombinant duplexes. Often these recombinant DNA molecules contain mismatched, or heterologous, base pairs: such molecules are called heteroduplex, or heterozygous, DNA molecules. See generally U. Goodenough, Genetics, 538-50 (3d Ed. 1984).
Homologous recombination is mediated by a protein called the recA protein In vitro, recA protein can induce a single stranded DNA fragment to invade a double stranded DNA segment and pair with a homologous region. The properties of recA protein, and the process of homologous recombination, are reviewed in Radding, C.M., Ann. Rev. Genet.16, 405 (1982).
The possibility for the integration of exogenous DNA into eukaryotic cells by homologous recombination has been the subject of recent investigations by several groups. Rauth, S. et al., Proc. Natl. Acad. Sci. USA 83, 5587 (1986) examined the ability of circular, single-stranded DNA to participate in a homologous recombination event in mammalian cells by inserting a fragment of the neomycin resistance gene into a single-stranded vector, mixing the vector with a double-stranded deletion derivative of pSV2neo, and testing the mixture for recombination in human cells, monkey cells, and nuclear extracts obtained from human cells. Recombinant molecules containing wildtype neomycin resistance genes, apparently resulting from a correction of the deletion in the double-stranded pSV2neo deletion derivative, were recovered from all three systems.
Thomas, K.R. et al., Cell 44, 419 (1986) describe a homologous recombination event between a gene residing in a eukaryotic cell chromosome and an exogenous gene introduced into the cell by microinjection. The chromosomal gene was a defective neomycin resistance gene which had been inserted into a plasmid, and the plasmid then inserted into the chromosome. The exogenous gene was similarly located in a plasmid.
Smithies, 0. et al., Nature 317, 230 (1985) describe an elegant procedure in which a homologous recombination event was used to insert an exogenous plasmid into a predetermined location in a mammalian chromosome, with the exogenous plasmids being introduced into the cells by electroporation. This work built in part on the work of Orr-Weaver, T.L. et al., Proc. Natl. Acad. Sci. USA 78, 6354 (1981), who accomplished the integration of a plasmid into a yeast chromosome through a homologous recombination event.
Electroporation, noted in connection with the Smithies work cited above, is a process in which brief electric impulses of high field strength are used to reversably permeabilize cellular membranes. The pores created during this process permit the introduction of macromolecules such as DNA. A drawback of electroporation, however, is that it reduces the viability of treated cells by 5 to 10 percent. A decrease of this size is a significant disadvantage in such procedures, where the quantities being manipulated are small, and where the materials used can be very expensive.
Other methods which have been used to introduce DNA into cells for various purposes have their own limitations. The most frequently used method, the uptake of calcium phosphate/DNA co-precipitates, is of limited utility for non-adherent cells. Other chemical methods, such as DEAE dextran-mediated uptake, are not suitable for stable gene expression. Protoplast treatment with polyethyleneglycol, as with any chemical method, is inherently cytotoxic. Biological methods, such as protoplast fusion, the use of liposomes, or the use of viral and retroviral vectors are restricted either to certain cell types or by the presence of potentially harmful DNA in the newly transformed cell. There is, accordingly, a need for a simple, reliable procedure by which an exogenous DNA sequence can be introduced into a cell, for exchange with a homologous DNA sequence in the cell. While additional background materials are introduced and discussed below, applicant is unaware of any suggestion in any of these materials of the unique combination of features which comprise the invention disclosed herein.